Composition of matter and methods for a fecal-derived sterilized prebiotic and postbiotic

ABSTRACT

Methods and compositions relating to sterilized fecal microbiota and compositions derived from the same. A method of sterilizing a fecal composition to obtain a sterilized fecal composition is provided. Methods of further purifying the sterilized composition and optionally freezing or freeze-drying the compositions is also provided. Methods of using the compositions for treatment in beverages, foods, and in supplements are also provided.

PRIORITY CLAIM

This application claims priority to U.S. provisional application No.62/923,174, filed Oct. 18, 2019, the content of which is incorporatedherein in its entirety by reference.

FIELD

This application the manufacture of compositions obtained from relatesto sterilized fecal microbiota transplantation (FMT) and fecal-derivedsterile postbiotic (FSP), the methods of making the compositions, andmethods of using the compositions.

BACKGROUND

For various medical reasons, it may be desirable to deliver healthyhuman stool and/or postbiotics extracted from stool to a recipient. Thedelivery of healthy human-derived stool to the colon of a recipientpatient is generally known as fecal microbiota transplantation,microbial transfer therapy, or fecal transplant. In recent years, thetransplant of healthy, live microbiota to a recipient has been usefulfor treating recurrent and/or antibiotic resistant Clostridium difficile(rCDI) infections.

This therapy has gained popularity and acceptance as a standard of carefor rCDI in the United States, Australia, and many parts of Europe. Insome cases, fecal transplant may be useful in treating patients havingrCDI infections that have not responded to other therapies, and/or whereother therapies (such as vancomycin, fidaxomicin, or metronidazole) maynot be suitable for a particular patient. For such patients, a livefecal transplant may safely and effectively restore the microbiota in apatient's colon to a healthy state.

Although agencies like the U.S. Food and Drug Administration (FDA) haveissued general guidance on safely performing fecal transplants, thereremains a lack of regulation for FMT manufacture. Additionally, FMT hasbeen linked with several safety concerns. Safety concerns regardinginfectious disease are particularly worrisome for immunocompromisedpatients (e.g., patients having AIDS, cancer, diabetes, certain geneticdisorders; patients who are malnourished). In addition, the transmissionof novel viral and parasitic pathogens, like SARS-CoV-2, through liveFMT transfer remain of serious concern. FMT remains with risks andpotential side effects for the patient receiving the transfer. Much, butnot all, of these risks relate to transmitting infection from the donorstool to the recipient.

Accordingly, a need has emerged for solutions that may help to maximizethe potential for the safe and effective treatment of illnesses, whileminimizing the potential for transmitting infection and other adverseside effects.

SUMMARY

The disclosure provided presents new methods of providing patients withthe benefit of FMT while reducing the risk of introducing otherillnesses. The present methods of making the compositions and systemstherefore, compositions derived and methods of using the compositionsrelated disclose systems and methods relating to sterilized fecalderived products also referred to as sterilized fecal-derived postbiotic(FSP).

A method is provided for making a sterilized fecal derived postbioticcomposition comprising: (a) blending a stool sample from a subject witha buffer; (b) removing particulate and fibrous matter from the blendedstool sample to obtain a liquid; and (c) sterilizing the liquid toproduce the sterilized fecal derived postbiotic composition. The methodcan further comprise a step of stabilizing the sterilized fecal derivedpostbiotic composition by lyophilization. The method can be performedwherein the stool sample is frozen when collected from the donor. Themethod can optionally adjust the pH of the fecal derived postbioticcomposition is adjusted to about 6.0 to about 9.0 prior tolyophilization. Another method contemplates obtaining stool from ahealthy screen donor. Donor screening can be performed using various ofthe criteria described herein in order to identify a healthy donor.

Another aspect of the disclosed method contemplates sterilizing byautoclaving. If the method used autoclaving, the step can be performedat about 121 to 134° C. for about 30 minutes or at about 135 to 140° C.for about 20 minutes.

The sterilization step of the methods described can be performed priorto blending the stool (step a) or after blending the stool or afterremoval of fiber and particulate.

The method of blending the stool of step (a) of the described method canbe accomplished by the use of a comminution device selected from thegroup consisting of: a crushing device, a grinding device, and ahomogenization device. The blending of the stool is performed in thepresence of the addition of water, a phosphate buffered saline solution,or maldextrin-trehalose solution.

In one embodiment, the method step of removing fiber and particulatematter can be accomplished by a method selected from the groupconsisting of: centrifugation, filtration, membrane filter press drying,or a combination thereof. The removing step can be a single step processor can involve one or more of these methods in sequence.

A product is produced by any of the described methods and can be used ina variety of manners. A sterilized fecal-derived post biotic compositioncan be obtained by the above described method. A sterilizedfecal-derived postbiotic composition comprises one or more of shortchain fatty acids, bile acids, amino acid derivatives, ceramides,lipopolysaccharides, capsular polysaccharides, and sphingosines. Theshort chain fatty acid of the sterilized fecal-derived postbioticcomposition can comprises formic acid or one of its salts and/or one ofits esters, acetic acid or one of its salts and/or one of its esters,propionic acid or one of its salts and/or one of its esters, butyricacid or one of its salts and/or one of its esters, isobutyric acid orone of its salts and/or one of its esters, valeric acid or one of itssalts and/or one of its esters, or isovaleric acid or one of its saltsand/or one of its esters. In another embodiment, the sterilizedfecal-derived postbiotic composition can comprise a ratio by weight ofacetic acid:propionic acid:butyric acid respectively of between 2:2:1and 3:1:1. Alternatively, the sterilized fecal-derived postbioticcomposition can comprise a ratio by weight of acetic acid:propionicacid:butyric acid respectively of 60±5:20±5:20±5.12.

The composition can be formulated to comprise a co-emulsifying agent,wherein the co-emulsifying agent is selected from the group consistingof: cetyl alcohol, stearyl alcohol, octacosanol, palmitic acid, stearicacid, and combinations thereof. In another embodiment, a sterilizedfecal-derived postbiotic composition can be formulated to furthercomprise at least one component selected from the group consisting of: ascreening agent, a vitamin, an essential oil, a plant protein, ananti-oxidizing agent, a preserving agent, a fragrance, a ceramide, amoisturizing agent, a lubricating agent, a polysaccharide, a filler andcombinations thereof. A formulated sterilized fecal-derived postbioticcomposition can be in the form of a solid, an aerosol, a pill, acapsule, a tablet, a paste, a powder, a gel, a lotion, a liquid, or abody wash.

In one embodiment, the sterilized fecal-derived postbiotic compositioncan be admixed or co-administered with a probiotic, a livebiotherapeutic, a synthetic microbial community, or a non-sterilizedfecal composition. Alternatively, the sterilized fecal-derivedpostbiotic composition can be used as a combination therapy with aprobiotic, a live biotherapeutic, a synthetic microbial community, or anon-sterilized fecal composition.

In another embodiment, a sterilized fecal-derived postbiotic compositioncan be used to treat or ameliorate a disease or condition in a subjectcomprising: administering to the subject (i) the sterilizedfecal-derived postbiotic composition obtained by any of the methodsdescribed above, or (ii) a sterilized fecal derived postbioticcomposition comprising one or more of short chain fatty acids, bileacids, amino acid derivatives, ceramides, and sphingosines in atherapeutically effective amount, wherein the condition is selected fromthe group consisting of a Clostridium difficile infection, C. difficilerelated condition, a mood disorder, fatigue, autism spectrum disorder(ASD), inflammatory bowel syndrome constipation (IBS-C), inflammatorybowel syndrome diarrhea (IBS-D), inflammatory bowel disease (IBD), asubject who has received immunotherapy to treat a cancer, a metabolicsyndrome, and dysbiosis. The mood disorder being treated or amelioratecan be anxiety, depression or obsessive-compulsive disorder.

In another embodiment, the method contemplates administering asterilized fecal derived postbiotic composition described above to thesubject with a non-sterilized fecal composition, live biotherapeutic,and/or a probiotic.

A method of treatment or amelioration contemplated includes oral,topical intranasal, rectal vaginal and intravenous administration of thesterilized fecal derived postbiotic composition.

Another embodiment contemplates using a sterilized fecal derivedpostbiotic composition as an additive in a culture media. The culturemedia can be for culturing a probiotic, a live biotherapeutic, or asynthetic microbial community.

A further embodiment contemplates adding a sterilized fecal derivedpostbiotic composition to a dietary supplement, a food, a food product,or a beverage.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures assist in illustrating aspects of the methods andcompositions described.

FIG. 1 provides a graph of extract components in fresh versus frozenstool samples, according to aspects of the present disclosure.

FIG. 2 provides a graph of the retention of SCFA throughout processingsteps, according to aspects of the present disclosure.

FIG. 3 provides a graph of the effect of adjusting pH beforelyophilization on the extract components.

FIG. 4 provides a box plot illustrating taurodeoxycholic acid relativeconcentration in FMT vs FSP from different donors.

FIG. 5 provides a box plot illustrating taurodeoxycholic acid relativeconcentration in FMT vs FSP vs FSP-L from the same donor.

FIG. 6 depicts a comparison of Altered Metabolites P<0.1 between an FMTProduct and a sterilized FSP Product as obtained using the method ofExample 1 when using fecal samples from the same donor or from differentdonors.

FIG. 7 depicts tryptophan metabolism in different donors as between aFMT product and a non-lyophilized FSP (sterilized fecal-derivedpostbiotic). The FMT again appears as the 3 dots at the bottom while theFSP appear above those with the lighter circles.

FIG. 8 depicts tryptophan metabolism of a fecal sample from the samedonor as between a FMT product and a FSP (sterilized fecal-derivedpostbiotic) and a lyophilized FSP (FSP-L). The FMT appears in the lower3 dots of the lower circle in FIG. 8 . The tryptophan degradation of theFSP-L has a scatter line appearing that ties the three dots together inthe upper circle in FIG. 8 .

DETAILED DESCRIPTION

Rather than focusing on live bacteria, the composition and method ofmaking the composition described herein involves a fecal derivedpostbiotic product (also referred to herein as “FSP”). A FSP compositionor extract is sterilized to exclude any live organisms (includingbacteria, archaea, fungi, parasite, or protozoa and viruses). Alsodescribed herein are methods of using sterilized FSP compositions.

1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present disclosure, the preferredmaterials and methods are described herein. The following terminologywill have the indicated meanings unless specifically indicatedotherwise.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element. Thus, recitation of “a cell”, for example, includes aplurality of the cells of the same type.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of +/−20% or +/−10%, or +/−5%, or +/−1%, and +/−0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

As used herein, the term “autologous” is meant to refer to fecalmaterial derived from the same individual to which it is later to bere-introduced into the individual. As used herein, “heterologous” isused to refer to fecal material obtained from one individual (a donor)and to be used a second individual.

An “effective amount” as used herein, means an amount that provides atherapeutic or prophylactic benefit.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and can include a human being.

To “treat” or to “ameliorate” a disease or a condition as the terms areused herein, means to reduce the frequency or severity of at least onesign or symptom of a disease or disorder experienced by a subject.

By “sterilizing” or “sterilization” is meant to include autoclaving,sonicating, UV-C light sterilization, ozone system sterilization,pasteurizing, tyndallization, and microwave irradiation. A preferredmethod of sterilization is by autoclaving. A standard temperature forautoclaving is 121° C. for 15 to 30 minutes with saturated steam. Apreferred autoclaving time is at about 121 to 134° C. for about 30minutes or at about 135 to 140° C. for about 20 minutes.

By “removal of particulate and fibrous matter” is meant to include theremoval of material from the fecal or stool sample. For example, aftercentrifuging a slurry in a 50 ml conical tube, about 5 to about 35 ml ofthe about 50 ml total slurry is discarded as fiber and large undesiredparticulate.

Ranges are provided throughout this disclosure; various aspects of thedisclosure can be presented in a range format. It should be understoodthat the description in range format is merely for convenience andbrevity and should not be construed as an inflexible limitation on thescope of the disclosure. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible subrangesas well as individual numerical values within that range. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed subranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5,5.3, and 6. This applies regardless of the breadth of the range.

The terms “fecal” and “stool” are used herein interchangeably. “Rawstool” or “raw fecal samples” indicate that the sample have not hadanything added to them or have been otherwise treated.

As used herein, a “postbiotic” is a sterile fecal-derived postbioticcomposition or product containing said composition that includesbacterial primary or secondary metabolites, cellular components of oneor more killed bacteria, viruses, fungi, archaea, food by products, anddonor-derived molecules. Short chain fatty acids (SCFAs) are an exampleof a primary metabolite.

As used herein, a “probiotic” can include live bacteria and live yeaststhat are beneficial to the subject to which they are administered.

As used herein, a “live biotherapeutic” or “LBT” is a biological productthat contains live microorganisms such as bacteria and/or yeast that arenaturally occurring, recombinant, or clonally selected and whenadministered to the subject confer a health benefit to the subject, asynthetic microbial community, or a non-sterilized fecal composition.Probiotics can be in the form of capsule as supplements or in foods,like yogurt. Lactobacillus spp. are examples of common probiotic speciesas are Bifidobacterium, and Saccharomyces boulardii.

As used herein a “synthetic community” and a “synthetic microbialcommunity” are the construction of a microbial system that is simplifiedand not found in nature.

As used herein, a “fecal microbiota transplantation (FMT)” is a processof transferring fecal material from one subject to another subject.Generally, the transplantation process uses non-autologous fecal matter.The fecal microbiota (or fecal transplant) is generally obtained from ahealthy, screened donor and is administered in to the colon of arecipient. The process can also be also referred to as microbialtransfer therapy, microbiota restoration therapy, intestinal microbiotatransfer, donor feces infusion, stool transplant, or fecalbacteriotherapy. As used for the compositions and methods describedherein, an FMT product is defined as a fecal pellet derived from ascreened donor stool that is processed into a concentrated bacterialpellet. A method for concentrating the bacterial pellet is known asdescribed for example in Borody 2012, Hamilton et al., “StandardizedFrozen Preparation for Transplantation of Fecal Microbiota for RecurrentClostridium Difficile Infection,” Amer. J. Gastroenterology 107(5):761-767: U.S. Pat. 10,226,431. In all instances, “FMT” and “fecalmicrobiota transplantation” indicate a sample that has not beensterilized and contains live bacteria.

2. Obtaining and Preparing Sterilized Fecal Derived Products

As with any medical procedure, there are both known and unknown risksassociated with unsterilized FMT. These risks are commonly associatedwith infectious organisms and/or substances in donor stool that were notidentified through screening. Such risks may be mitigated, but cannot becompletely eliminated, even by extensively screening donors prior tostool collection to minimize the risk of exposing the recipient to aknown infectious disease or other risk factors.

For the sterilized fecal-derived postbiotic described herein, potentialdonors may be screened for their complete medical history and receive atleast one physical exam by a qualified physician. Generally, thispre-donation screening may help ascertain that a potential donor is freefrom the following non-exhaustive list of afflictions: chronic disease,autoimmune disease, current or past chronic gastrointestinal disease,atopic asthma, atopic dermatitis, diabetes, metabolic syndrome, mooddisorders, chronic pain, and/or infectious diseases. See also thepathogen table infra. Mood disorders considered for treatment oramelioration of a condition include anxiety, depression andobsessive-compulsive disorder.

While unsterilized FMT derived products contain live bacteria, theproducts and compositions described herein do not. The sterilizedproducts, extracts and components derived from the products provide aliving-organism free composition that is safe for administration. In oneembodiment, it is contemplated that the sterilized fecal derivedpostbiotic composition described herein can be used as an adjuvant inconjunction with non-sterilized FMT products containing living organismsor synthetic communities of gastrointestinal associated bacteria. Thecompositions described herein can also be used as a stand-alonecomposition for administration to treat or ameliorate a condition or incombination with other therapeutics.

The sterilized fecal-derived postbiotic composition described herein areinherently safer because they eliminate infectious disease risk. This istrue for both oral ingestion of the material or topical application.Stool contains bacteria that can overgrow. This overgrowth can causeimbalance in both the gut and on the skin. Bacterial overgrowth on theskin, for example, can cause a topical infection. Sterilization of thefecal-derived postbiotic composition, such as via autoclaving,definitively kill all bacteria and viruses while retaining bacterialmetabolites.

Non-sterilized FMT products is developing a growing body of researchsuggesting short-term and some long-term safety based on treatment datafrom patients with rCDI and IND clinical studies (Perler, 2020).Non-sterile FMT compositions have been used with over 50,000 patients inthe U.S. since 2012 when OpenBiome became the first national stool bank(Panchal 2018). The most commonly reported side effects from non-sterileFMT administration observed in treated patients are diarrhea, cramping,and bloating that are minor in nature and often resolve followingtreatment (Hefazi 2017, Quraishi 2017, Iqbal 2018).

Large scale clinical research specifically designed to address safetyand tolerability of non-sterile FMT in healthy populations has beenlacking. This may be in part due to the financial burden of thesestudies and to the speed with which the non-sterile fecal transplant wasintroduced. However, the noticeable efficacy of non-sterile FMT intreating Clostridium difficile infections and related conditions allowednon-sterile FMT to propel forward in a non-traditional drug pipeline asan investigational new drug (IND) for disease states. Prior to 2019, allsevere side effects reported were related to delivery routecomplications or co-morbidity complications in patients treated withnon-sterile FMT (Baxter 2016). In 2019, the first death attributeddirectly to non-sterile FMT occurred in an IND trial withimmunocompromised participants where a previously unidentified multidrugresistant organism created septicemia and death as reported by the USFDA in June 2019. This further elucidates the need for a safer optionfree of infectious disease risk.

Infectious disease risk remains the most serious safety concern withnon-sterilized FMT as a therapeutic tool. Fecal-oral transmission is acommon evolutionary tool for many infectious pathogens to exude theirphysiologic effect (de Graaf 2017). Further complicating this risk isthe potential for stool donors for the non-sterile product to beasymptomatic carriers of infectious pathogens. In the coronaviruspandemic, asymptomatic carriage and transmission of SARS-CoV-2 throughstool is a very serious theoretical concern (Nicco 2020). In addition toSARS-Cov-2, another concern for infectious disease transmission ismulti-drug resistant organisms (MDROs) especially in immunocompromisedpatients (Wardill 2019). There have been several clinical studies andcase reports supporting safety and efficacy of non-sterile FMT treatmentin these immunocompromised patients (Shogbesan 2018, Mandalia 2016,Kelly 2014, Limketkai 2019, Di Bella 2015). However, in 2019 twopatients developed extended-spectrum beta-lactamase (ESBL)—producingEscherichia coli bacteremia and one died. These two patients were linkedto a single donor who was an asymptomatic carrier of this MDRO(Jorgensen 2017). In addition to MDROs, there remains a concern relatedto asymptomatic donors with other potential pathogens including C.difficile, giardia, and norovirus (Paramsothy 2015, Schwartz 2013).Although these pathogens mentioned can be tested for in the donor stoolfor use in preparing non-sterile FMT and are outlined in many of theconsensus screening protocols, there is no way to screen for unknownpathogens that are yet to be identified (Cammarota 2019, Haifer 2020).

Sterilized Stool Postbiotic Processing. In some embodiments, the methodsdescribed herein may include the placing of healthy donor stool in anautoclaved container suitable for blending. In some embodiments, anautoclaved buffer solution or water may be added to the container,depending on the condition of the donor stool. The stool and buffersolution (or water) may be blended together to produce a fecal slurry.This method may include adding a buffered solution of PBS,maldextrin-trehalose solution, or deionized water (DI H₂O) before,during, and/or after the homogenization. The homogenized slurry can beproduced in a blender or using another suitable homogenization method,including but not limited to mashing in a bag. The method may nextinclude the step of filtering bulk fiber and large particulates from thehomogenized mixture. Separation of the large particulate and fiber mayoccur through physical filtration using filter paper or mesh straining.Alternatively, centrifugation can be used for filtration.

If centrifugation is used to prepare the sterilized fecal derivedpostbiotic, then the fecal slurry is poured into sterile vials or tubessuitable for use in a centrifuge and undergo at least one centrifugationcycle. Next, the vials may be removed from the centrifuge so that thefiber and large particulate pellet may be discarded. The supernatantfrom the at least first cycle may be poured into new sterile containers.

In other embodiments, the resulting fecal slurry after blending may befiltered using another filtration process that is not centrifugation,but rather physical filtration using filter paper or mesh straining.Further processing steps may include the concentration of thesupernatant into a bacterial pellet with the option of additionalcentrifugation. The bacterial pellet obtained after removal of theinitial fiber and large particulate removal may be resuspended, forexample, at a 1:1 ratio with the fecal supernatant.

Next, the bacterial pellet, supernatant or filtered slurry may besterilized. In some embodiments, sterilization may be performed usingany of the following non-exhaustive means of sterilization: UV-C light,ozone, sonication, microwave irradiation, pasteurization, autoclaving,tyndallization, or any other suitable sterilization technique. In someembodiments, the sterilized fecal product may then be stabilized using afreeze-drying technique also known as lyophilization or cryodesiccation.The pH of the product may be checked prior to the freeze-dryingtechnique, and if the pH is below 6.0, NaOH may be added to increase thepH from about 6 to about 9.0, or higher than 6.5, or 7.0, or 7.5 or 8.0.Unexpectedly, by increasing the pH at this stage, improves the abilityto maintain short chain fatty acid metabolites of the sterilizedfecal-derived postbiotic during lyophilizatoin. Lyophilization orfreeze-drying allows the sterilized fecal product to be preserved in acondensed dry powder that is stable for storage and use at roomtemperature. The lyophilized sterilized fecal-derived postbiotic powdermay then be used for oral, topical, vaginal or rectal intake, intranasaladministration, or other formulations for intravenous or parenteraladministration as contemplated herein.

In some embodiments, the method may include freezing the stoolimmediately after collection. Freezing of the stool can be done at −6°C. to −80° C. The ability to freeze, store, and then transport to thenthaw for manufacturing allows for various advantages. Traditionally,non-sterile FMT is processed within 12-24 hours of donation with freshstool. When the live bacteria are the focus of the end-product, thisrepresents an obvious requirement. A final product of a sterilizedfecal-derived postbiotic composition and its metabolite components asdescribed herein provides a benefit, because initial freezing of a stoolsample does not impact the final composition and significantly improvesthe efficiency of the process. A review article investigating change inmetabolomic profiles using various manipulations such as process delays,and temperature elucidated this point. It is known that one to twofreeze/thaw cycles are insignificant when assessing change in metaboliteprofiles for serum or blood (Stevens 2019). Likewise, frozen or freshstool from the same donor maintains a similar metabolite profile forSCFA (FIG. 1 ). Further, by having donors freeze multiple stools athome, not only does this increase convenience in bulk pick-ups, but alsoallows a greater donor population geographically. Donors will no longerbe limited by location. This method provides a decentralization of thestool banks with the capacity for reduced human interaction. In someembodiments, stool samples can be collected, frozen, and couriered ondry ice to a processing location.

In an embodiment, the method of processing stool can comprise thefollowing steps for developing a sterilized fecal-derived postbioticcapsule.

Ingredients:

-   -   One previously frozen complete bowel movement rated as Bristol        3-4 only    -   Distilled water

Procedure:

-   -   1. Stool collection and transport        -   Stool is collected by donor            -   Stool is immediately frozen in a dedicated freezer            -   Stool is transported or shipped in a cooler with dry ice    -   2. Stool processing        -   i. Record the stool donor identification, total stool            volume, Bristol stool scale for donation, time of stool            donation, and date of donation.        -   ii. Frozen stool is removed from the freezer and allowed to            thaw for up to 24 hours in the refrigerator.        -   iii. Measure out 50-150 g portions of stool into an            autoclaved blending jar. Record gram amount of stool.        -   iv. Add distilled (DI) water in a 5:1 ratio based on stool            mass to the blending jar (ex: 500 mL DI water to 100 g of            stool). Record volume of DI used.        -   v. Blend stool and distilled water for 60 seconds.        -   vi. Pour 50 mL of slurry into single-use 50 mL centrifuge            conical tubes. Record total number of conicals used.        -   vii. Place conicals in centrifuge for 1^(st) run—15-20            minutes at 2000 RPM (872G).            -   This step can be repeated if a particular donor's stool                is found to be extra fibrous.        -   viii. Remove conicals from centrifuge and pour supernatant            into new sterile 50 mL conicals. Record estimated volume of            fiber pellet.        -   ix. Discard fiber pellet with 50% bleach added to each            conical.        -   x. Place new supernatant filled conicals in centrifuge for            2^(nd) run for about 25-30 minutes at 4600 RPM 4613 G.        -   xi. Remove conicals from the centrifuge and pour off            supernatant into a sterile glass container. Keep the            supernatant for later use.        -   xii. Resuspend and homogenize pellets using a vortex and            transfer pipette. Pool the pellet in 1-2 50 ml conicals.            Record total volume of microbial pellet.        -   xiii. Resuspend total pellet 1:1 with previously saved            supernatant.        -   xiv. Pour the above pellet:supernatant mixture into a glass            jar, add a self-sealing lid and label with autoclave tape.            Record donor ID and date of stool donation on the autoclave            tape.        -   xv. Autoclave for 30 minutes at 121° C.        -   xvi. Remove from the autoclave and verify autoclave tape            shows completion. Allow the batch to cool on the counter and            place it in the refrigerator for overnight storage.        -   xvii. Take sterile product out of the refrigerator after it            has cooled.        -   xviii. Triple encapsulate 0.5 mL liquid product obtained for            a frozen non-lyophilized FSP product        -   xix. Alternatively, measure and adjust pH to 7.2 or higher            if needed using NaOH        -   xx. Pour into autoclaved lyophilizing glassware. Record            total volume/weight.        -   xxi. Flash freeze in a 90% isopropyl alcohol dry ice bath.        -   xxii. Lyophilize for 12-24 hours at <0.3 mBarr        -   xxiii. Remove sterilized fecal-derived probiotic from            lyophilizer, weigh the dry sample. Record total dry weight.        -   xxiv. Place powder in an autoclaved jar for storage. Remove            from glassware and store in 50 mL sterile conical at −20° C.            to reduce moisture exposure (can be stored at room            temperature).

This section describes general features of systems and methods relatingto sterile fecal derived probiotic compositions. In one embodiment, themethod of production includes collecting stool from a screened donor. Asecond step may then be to process and filter the collected stool. Athird step may then be to sterilize the microbial pellet using anautoclave, sonicator, UV-C light, ozone system, microwave irradiation,or other suitable sterilization technique. A fourth step may then be tofreeze dry or lyophilize the pellet. The order of sterilization andfreezing can be reversed. A fifth step may be to verify sterility, suchas by using some form of a live/dead bacterial staining and otherculturing techniques capable of detecting germination of spores forexample. This method of manufacture produces a sterile freeze-driedfecal derived postbiotic powder that can then be subsequently orally,topically, vaginally, rectally, or intranasally delivered to arecipient—or stored for later use.

Specific protocols for each stage of FSP therapy are discussed herein,from donor recruitment, to sterilization, to outcome reporting. Thesestages may include the following, without limitation: recruitingpotential donors, rigorous screening of potential donors, selectingqualified donors, providing stool donor instructions, transporting stoolsamples, receiving said samples in a laboratory, producing capsules orproduct, tracking, delivery, and outcome reporting. Of course, not allembodiments will include each stage, and certain stages may be completedindependently of others. Ultimately, the objective of the presentteachings is to increase the likelihood of successful treatment or useof the sterile product, and to minimize the risk of transmitting diseaseor other risk factors to the recipient of the therapy or product.

Donor Identification. A suitable stool donor can be identified using afecal transplant questionnaire. If the responses meet qualificationsadditional screening is performed. Additional screening can includequestioning the potential donor in an interview regarding the answers tothe questionnaire, obtaining basic health history and health practices.

Donor screening. If the potential donor satisfies this set of testing,then the potential donor may receive a physical examination, is screenedfor any presence of infectious disease symptoms and provides a stool andserum sample for analysis. Potential donor's serum can be obtained toassess for the presence of an HIV strain or for syphilis for example.HIV and syphilis can be retested after 3 weeks from the initial testingto check and confirm the absence of seroconversion. All serum and stoollaboratory testing can be performed by a qualified laboratory.Additionally, stool analysis can include analysis to detect the presenceof 6 Multi-Drug Resistant Organism genes. Testing can be performed in astep-wise manner. In one aspect, stool assessment is done in less than14 days. Upon receipt of assessment, if deemed qualified, potentialdonors are further screened based on a serum sample and providedperianal swabs for both MRSA and CRE. Serum and stool testing can beperformed using a certified laboratory. Additional seroconversionstudies for the presence of human immunodeficiency virus (HIV) andsyphilis are performed three weeks after initial screening and beforerelease of a donor's fecal material. Donor stool is not used for capsuleproduction until this information is obtained and confirmed.Comprehensive analysis of the stool is also performed to assess quality.

Recruitment of a human donor can begin with a short online surveytargeting health conscious communities. Key aspects of this surveyidentify eligibility. Donors are considered eligible when vaginallybirthed and breastfed exclusively for 6 or more months. Additionally, apotential donor cannot have been diagnosed with a pre-existingcondition. A potential donor is interviewed regarding the donor's dietand lifestyle. Donor screening takes into consideration food choices, adonor's physical activity, and their mental health including a donor'sstress management ability, and community.

In the event a qualified donor remains active, then the donor will beasked to undergo selected tests every 6 months or earlier depending on anew exposure or illness.

Donor recruitment generally describes the process of selectingindividuals who may be of suitable health and lifestyle for donatingstool. During the steps of donor recruitment, screening, and selection,the overall health of the donor is critically important to the safetyand efficacy of the product. By administering health tests and carefullyassessing the donor's microbiome history, a physician or other FMTservice provider may be able to minimize the potential for diseasetransmission. In some embodiments, the initial donor recruitment stepmay include a simple intake questionnaire, which—if passed—may lead intoa more comprehensive questionnaire if the potential donor continuesthrough the recruitment process. An optimal donor questionnaire will aidthe FMT service provider in ensuring the overall physical, mental, andemotional health of the donor—all of which may impact the bacterialdiversity of the potential donor.

Although donors may be recruited in any number of ways, it may bedesirable—in some embodiments—to recruit donors from a target group ofindividuals who may be more likely than the general population tosatisfy certain requirements for suitable donors. For example, in someembodiments, donors may be recruited from a group of individuals whoengage in outdoor activities, eat organic foods, and/or generally live ahealthy lifestyle. For some donors, certain health and lifestyle factorsmay have a positive impact on the donors' microbiome diversity andthereby increase the likelihood of successful treatment for the productrecipient. An example of such a target group may include, for example,members of communities in Blue Zones, naturopathic doctors, athletes,and so on—who may have healthier habits than the general population.However, selecting donors from such target groups is not necessary, assuitable donors may exist in any demographic, so long as such donors areproperly screened and examined by a qualified FMT practitioner.

Because research regarding the microbiome is ongoing, it may bedesirable to approach donor selection from a holistic perspective inorder to maximize the likelihood of a safe and successful FMT derivedproduct. Rigorous donor screening may thus include acquiring a detailedaccount of a potential donor's travel history, antibiotic exposure,pharmaceutical medications, known allergies, personal or family historyof cancer, gastrointestinal disease, autoimmune conditions, and otherfactors that may exclude an individual from becoming a stool donor.Additionally, diet and lifestyle factors may also be considered whenselecting donors. In some embodiments, an ideal donor may eat a healthy,well-balanced diet (such as by increasing intake of varied, organicfruits and vegetables while minimizing intake of processed and/orunhealthy foods) and generally engage in healthful practices thatsupport holistic wellbeing of the individual.

Stool sample assessment. Stool is analyzed again through screening.Screening questions include, without limitation: frequency of bowelmovements per day/week, how those bowel movements typically rate on theBristol stool scale, dietary history, relationship with food, whetherthe potential donor was born vaginally and/or breast-fed, theenvironment the potential donor was raised in, number and age ofsibling(s), exposure to environmental contaminants, treatment forcertain types of infections, history of antibiotic use, weightfluctuations, family health history, characteristics about the potentialdonor's menstrual cycle (if applicable), age, occupation, weight,height, exercise habits, smoking habits, drinking habits, sexualhistory, exposure to STIs, illicit drug use, recent tattoo/piercinghistory, hospitalization, surgery history, medical diagnosis history,mental health, allergies, international travel, prescription and/orsupplement use, microbiome history, and more.

Exemplary fecal/stool donor screening protocols can include thefollowing as well as modifications thereof.

Exemplary Donor Questionnaire

Microbiome History

-   -   How often do you have a bowel movement each week?    -   Where do your bowel movements typically rate on the Bristol        Stool Scale?    -   Please describe your dietary history from infancy. (e.g., vegan,        SAD, vegetarian)    -   Do you have a healthy relationship with food? Any history of        disordered eating?    -   Were you born vaginally?    -   Were you breastfed when you were a baby? Exclusively or with        formula supplement?    -   What kind of environment were you raised in? Urban, rural, etc.?        Exposure to dirt?    -   When have you had pets in your life? What type and what ages        were you?    -   Do you have any siblings? If yes, what is the age difference?    -   Were disinfectants used in your home often as a child?    -   Have you ever been exposed to any major environmental exposures,        defined roughly as chronic or acute exposure to: pesticides,        mold, lead, or other heavy metals, or contaminated water?    -   Have you ever been treated for an infection such as pneumonia or        a UTI that required antibiotics (Abx)? Ear infections or strep        throat as a kid? What kind of Abx?    -   How many times have you used antibiotics in your life?    -   When was the last time that you used antibiotics?    -   Have you ever experienced large changes in your weight over        short periods of time? Have you ever had a BMI over 30? Have you        struggled with weight loss or yo-yo dieting?    -   Have any members of your immediate family (parents, siblings)        been diagnosed with any medical conditions? If yes, please        elaborate on when and what conditions they have been diagnosed        with.

For persons with a vagina:

-   -   Do you have a menstrual cycle?    -   Is your cycle the same length each cycle? How many days?    -   Is your cycle light or heavy?    -   Do you have any PMS symptoms?

Social History

-   -   What is your age?    -   What is your occupation?    -   What is your current diet? Describe.    -   How often do you exercise? What kinds?    -   What is your current height and weight?    -   Do you smoke cigarettes?    -   Have you ever smoked cigarettes? When and how many?    -   Do you drink?    -   How many drinks a week on average do you consume?    -   Have you ever in the past considered your drinking habits to be        a problem?    -   Are you sexually active?    -   What are you sexual practices? Sex with men women or both?    -   Do you have more than one regular sexual partner?    -   Do you get screened for HIV, GC/CT, and syphilis between        unprotected sexual partners?    -   When was your last unprotected sexual event out of a monogamous        relationship?    -   Do you have any Sexually Transmitted Infections?    -   Have you ever been diagnosed with any Sexually Transmitted        Infections?    -   Have you ever been exposed to hepatitis A, B or C?    -   Have you been exposed to HIV?    -   Have you ever had sex for money or drugs?    -   Have you ever used IV illicit drug?    -   Have you had a tattoo or piercing in the last 6 months?    -   Have you ever been incarcerated?

Medical History

-   -   Have you ever been hospitalized? If yes, for what reason(s)?    -   Have you ever had any surgeries? If yes, what for?    -   Have you ever had any medical diagnoses from a physician?        -   Have you ever been diagnosed with any autoimmune conditions?        -   Have you ever been diagnosed with any chronic pain            conditions?        -   Have you ever been diagnosed with any metabolic conditions?        -   Have you ever been diagnosed with any neurological            conditions?        -   Have you ever been diagnosed with any psychiatric conditions            (e.g. major depression, generalized anxiety, bipolar, OCD)?        -   Have you ever had an atopic disease (e.g., asthma, atopic            dermatitis, eosinophilic disorders of the gastrointestinal            tract)?            -   Have you ever had any gastrointestinal conditions (e.g.,                history of IBD, IBS, chronic constipation, chronic                diarrhea, Celiac disease)?    -   Have you ever had a functional diagnosis? (e.g., adrenal        fatigue, hypothyroid, anemia, interstitial cystitis (IC),        fibromyalgia, chronic fatigue)    -   How would you define your mental health currently, in the past?    -   Do you or have you ever had any allergies, if so, please        describe when you developed them and what you are allergies.    -   Have you ever traveled outside of the country? If yes, when,        where, and for how long? Did you have gastrointestinal (GI)        symptoms while traveling?    -   Have you ever taken any pharmaceuticals? If yes, what and when?    -   What medications do you currently take including over the        counter?    -   Do you take any supplements? If yes, which ones and why?    -   Have you donated blood, platelets or plasma in the last 8 weeks?    -   Had any vaccinations or other shots in the last 8 weeks?

Donors can be screened for one or more of the following pathogens or aspecies falling within the following table, although this list is notmeant to be limiting.

Pathogen Table Viruses and Fungi Antibiotic Bacterial ParasitesPathogens Resistance Genes Clostridium difficile Necator americanusAdenovirus F40/41 Helicobacter - toxin A Clarithromycin Clostridiumdifficile Cryptosporidium Rotavirus A Helicobacter - toxin BFluoroquinolones Campylobacter spp. Entamoeba Astrovirus UniversalMicrobiota histolytica Resistance Genes - b-lactamase CampylobacterGiardia lamblia Norovirus GI Universal Microbiota jejuni ResistanceGenes - Fluoroquinolones Campylobacter coli Cyclospora Norovirus GIIUniversal Microbiota cayetanensis Resistance Genes - MacrolidesCampylobacter Chilomastix mesnili Sapovirus I Universal Microbiotaupsaliensis Resistance Genes - Vancomycin Plesiomonas Cyclospora spp.Sapovirus I shigelloides Vibrio Dientamoeba fragilis Sapovirus II(parahaemolyticus) Enteropathogenic Endolimax nana Sapovirus III E. coli(EPEC) Enterotoxigenic Entamoeba coli Sapovirus IV E. coli (ETEC)Lt/StE. coli O157 Pentatrichomonas Sapovirus V hominis Shiga-Like ToxinLarval Nematode Cytomegolovirus Producing E. coli (STEC) Shigella/EIECAscaris lumbricoides Epstein Barr Virus Heliobacter pylori Strongyoidsstercoralis Hepatitis A Listeria Taenia solim Hepatitis B Vibrio(cholerae) Schistosoma Hepatitis C Enteroaggregative Blastocystishominis HIV E. coli (EAEC) Klebsiella Isospora belli Candida albicanspneumonia Edwardsiella tarda Fasciola/ Candida spp. FasciolopsisYersinia Hymenolepsis Geotrichum spp. enterocolitica Vibrio (vulnificus)Dipylidium caninum Rodotorula spp. Salmonella Diphyllobothrium datumCryptosporidium Trichuris trichina Treponema pallidum Enterobiusvermicularis Mansonella Microsporidium spp. Ancylostoma duodenaleTrichuris trichiura Taenia spp.

Another screening example can include the following: phase 1 of thescreening (initial screening of the donor) can include an assessment ofthe donor's serum involving a CBC (complete blood count) withdifferential, a CMP (comprehensive metabolic panel), a HgA1C (hemoglobinA1C), and antibodies for HIV one and two (HIV-1 and HIV-2). Phase I forscreening the stool can include 16s diversity index sequencing,obtaining a metabolomics profile, O&P with giardia antigen, andculturing the stool for the presence of Salmonella, Shigella,Campylobacter, and EHEC.

Phase 2 of the exemplary screening can occur approximately every sixmonths or after new onset symptoms or a change in risk factors for thedonor occurs. Serum at this stage would be tested for CBC withdifferential and CMP.

A donor would also undergo a physical exam. An exemplary physical examchecklist can include one or more of the following: (1) Capillaryrefill, assess for clubbing and peripheral cyanosis; (2) Assessment ofCranial Nerves 2-12 (PERRLA, EOM, facial movements, tongue/palate,whispered voice test and trapezius shrug); (3) Inspection ofconjunctiva, lips and buccal mucosa; (4) Inspection of neck forappearance, symmetry, trachea position, and masses; (5) Palpation of thethyroid for enlargement, tenderness and masses; (6) Visual assessment ofrespiratory effort and signs of central cyanosis; (7) Lung auscultation;(8) Carotid auscultation; (9) Cardiac auscultation and palpation(reclined position); (10) Inspect the abdomen—assess for shape,distention, ascites, lesions; (11) Auscultate for renal, abdominalaortic, and iliac bruits, and bowels sounds; (12) Palpate the abdomenassessing for guarding, rigidity, tenderness and liver edge; (13) Assessfor edema of the lower extremities; and (14) Upper and lower extremityreflexes.

Such screening and testing of donors may include inquiries into recent(e.g., within 90 days) antibiotic use, which may exclude an individualfrom being a suitable donor. Other factors that may exclude anindividual from becoming a donor may include, without limitation:illicit drug use, intravenous drug use, recent tattoos and/or bodypiercings, sexual contact with someone who has tested positive for HIV,etc. Potential donors may also be screened for metabolic syndrome,autoimmunity (e.g., multiple sclerosis, rheumatoid arthritis, etc.),neurologic disease, neuropsychiatric syndrome, atopic disease fromrespiratory to skin reactions, malignancy, ongoing oncologic therapy,and/or chronic pain syndromes.

Although the mental health and well-adjustedness of a potential donormay be difficult to measure quantitatively, it may be possible to obtaina good sense of the suitability of a potential donor through carefulscreening of health, personality, and temperament, in addition to askingcomprehensive intake questions, performing infectious disease testing,and performing physical examinations. This extensive vetting process mayallow a physician to identify potential risk factors associated with apotential donor's lifestyle and overall health. In some embodiments,initial donor screening may be conducted by phone interview to coverbasic health history and health practices. If a potential donor passesthe phone interview, then a next step in the vetting process may be tofollow up with an in-person or remote video interview (e.g., telehealthconsult) to review and confirm the potential donor's answers to theintake questionnaire. A next step may then include screening thepotential donor for infectious disease, followed by a further step ofperforming a physical exam on the potential donor, or having them obtainone from their local physician if a remote donor.

In some embodiments, laboratory testing of potential donors during thescreening stage may include performing serum and stool tests. Thepathogen table herein includes an exemplary and non-exhaustive list oflaboratory tests that may be performed in order to screen individualsfor their suitability as donors. Generally, donations may not beaccepted if an otherwise qualified donor has experienced febrile,systemic, or gastrointestinal symptoms. In some embodiments, personal orfamily history that shows a propensity for IBD (inflammatory boweldisease), IBS (inflammatory bowel syndrome), and/or certain hereditarycancer may preclude an individual from becoming a donor due to currentlyquestionable immunological influence that may or may not be transmittedvia microbial information. There may be similar correlations between themicrobiome and factors such as obesity, metabolic syndrome, allergies,and autoimmune illnesses, such that it may be safer for the recipient ifthe FMT service provider decides to exclude potential donors based onthese factors.

In some cases, it may be desirable to perform these tests in multiplephases, based on common disqualifiers for potential donors. When phasesare used, donors who pass a certain phase of testing may then move ontothe next phase of testing. For example, Phase I may entail initialtesting for common stool pathogens. If the potential donor testsnegative for those, then said donor may move onto Phase II of testing.Phase II may include, for example, testing for: HIV, hepatitis A/B/C,syphilis, and/or others. If a donor passes Phase II of stool and serumtesting, then said donor may move onto Phase III, which—in someembodiments—may include testing for HIV and syphilis approximately three(3) weeks after Phase I of testing. If the donor passes Phase III, thensaid donor may move onto Phase IV, which may entail routine maintenancethat is performed approximately every six (6) months and/or as neededfor acute exposure or change in risk factors. Phase IV may include, forexample, testing for CBC, CMP, HIV, hepatitis C, syphilis, Giardia,Salmonella, Shigella, and/or Campylobacter.

These phases may or may not immediately follow each other, and phasesmay be modified as needed. Generally, the frequency and timing oftesting will vary depending on the illness or exposure being tested for.HIV and syphilis, for instance, may be re-tested several weeks after theinitial test to check for seroconversion. Follow-up testing forhepatitis B and C may also be useful. No donor stool should be used forpowder and/or capsule production until all testing information iscomplete and confirmed. Some tests may also be repeated every six (6)months throughout the donation time period as an extra safetyprecaution. Testing may be optional given sterilization of thesterilized fecal-derived postbiotic composition.

Exemplary donor intake questions can be modified. For example, insituations where a patient requests their intimate partner to be theirdonor, some of the tests for sexually transmittable infections may benulled due to the assumed passage of bodily fluids between the donor andrecipient. As another example, severely immunocompromised patients mayrequire donors who meet even more stringent criteria, such as those whoscreen negative for Epstein Barr virus or cytomegalovirus, andmulti-drug resistance organisms. Overall, donor exclusion criteria arevast, and the list of laboratory tests performed may also change as moreinformation becomes known about internal and external factors thataffect the microbiome.

Approved Donor Instructions. Once a donor is approved, said donor mayreceive a set of detailed donor instructions. The instructions mayinclude recommendations for maintaining a healthy microbiome throughdiet and lifestyle practices. For example, a donor may be instructed toeat an organic, whole foods-based diet that consists of varied fruitsand vegetables, and to drink enough water to stay hydrated. A donoroptionally may be instructed to engage in body movement each day, whichmay take the form of housework, gardening, walking, biking, etc. Suchmovement may be useful in maintaining a healthy microbiome in the donor.

Donor instructions may include symptoms that the donor must report aspost-screening approval. For example, the donor may be instructed toimmediately notify the FMT service provider if the donor experiences anychange in bowel habits; sign of cold, flu, or fever; change associatedwith risk of HIV or hepatitis contraction (e.g., change in sexualpractices, blood transfusion, needle stick incidence, new tattoos); useof antibiotics, etc. Further exemplifying the importance of thephysician-donor relationship.

In some embodiments, the donor instructions may also provide informationregarding which types of stool samples are acceptable based on theBristol Stool Scale or any other suitable stool chart, as well as otherinformation on safe collection, handling, and delivery of stool samples.This may be particularly useful where donors may be responsible forcapturing their own stool. For example, the donor may be instructed toplace a new stool specimen collector hat in a toilet, such as, but notlimited to, a Covidien Commode Specimen Collector or other suchcontainer that may be immediately sealed airtight and then refrigeratedto preserve bacterial diversity. The donor may also be instructed toavoid contamination of stool with urine, and to notate where thecollected stool falls on the Bristol stool scale, along with the dateand time of collection.

Sterilized Fecal Composition Tracking. Optionally, once the sterilizedfecal product is prepared, a tracking protocol can be implemented. Forexample, a processed stool sample can be given a batch ID number. The IDnumber may connect each product with its specific processing date,contained in a written database that includes important information,such as, but not limited to: the date and time the stool was collectedand processed, total volumes of material used, identity of the donor andthe processing technician, the specific gram/capsule measurements, andso on. Optionally, the tracking can also comprise a full log of suchinformation for safety and quality control. Additionally, in someembodiments, at least one sterilized fecal composition product from eachbatch may be saved for quality control tracking.

Stool Processing for Encapsulation. Once a donor is selected, stool iscollected and can undergo freezing, thawing, sterilization, and then befreeze-dried such that they are suitable for oral consumption by theirintended recipients. Product technicians may use protective gear (e.g.,gloves, face mask, goggles, laboratory coat, etc.) in a dedicatedworking space with a biologic refrigerator and freezer that are suitablefor processing product. Additionally, product handling may occur under abiological or chemical air-handling hood rather than in ambient air. Thepresent disclosure relates to fecal-derived sterile product manufacturedunder such conditions.

An exemplary protocol for the initial preparation of fecal-derivedsterile product can include: at least one fresh, complete bowelmovement; approximately 500-700 mL of sterile buffer solution. The stoolcan optionally contain up to 5 mL sterile cryoprotectant. Preferably, nocryoprotectant is required as all organisms are sought to be killedmaking a cryoprotectant unnecessary and unwanted. A stool that isconsidered a “3” or “4” on the Bristol stool scale may be used. In someembodiments, this donor stool may be collected by the donor in acontainer such as a Covidien Commode Specimen Collector and immediatelyrefrigerated. The donor stool may then be transported at a lowtemperature, such as in a cooler with ice packs, to a refrigerator at areceiving lab. A donor stool can be processed 1-3 weeks, 3 days, 48hours and within 24 hours of initial collection by the donor.

A preliminary step can include weighing the stool. For example, 100 g ofdonor stool may be weighed out and placed in a sterile containersuitable in which to blend the stool with the chosen buffer solution.The container may have a blender attachment, which may be used to blendthe donor stool with the buffer solution, such as saline, water, etc. Byway of illustration and not limitation, the container may be a 32-ouncesmall-mouth mason jar having a metal blender attachment to blend thedonor stool with DI water or a 1× phosphate buffered saline (PBS). Anysuch jar or other suitable container and blade should be autoclavable orsuitable for any other effective method of sanitizing equipment tocontrol for infection via sterilization.

Effective ratios of stool to buffer solution may vary, and such variancemay depend on the how the stool falls on the Bristol stool scale (or anyother such classification system for stool). For example, a blend ofapproximately 100 g of stool with approximately 500 mL of PBS. This 100g: 500 mL ratio may be suitable for donor stool that is considered to bea “4” on the Bristol stool scale. Other ratios may be more suitable fordonor stool that falls elsewhere on the Bristol stool scale. Forinstance, it may be suitable to add approximately 700 mL of buffersolution to 100 g of donor stool that is classified as a “3” on theBristol stool scale. In other words, the drier the donor stool is, themore buffer solution that may need to be added to the donor stool. TheFMT technician may have some discretion here, so long as ratio of donorstool to buffer solution is reasonably calculated to produce asemiliquid mixture—or a slurry—after blending.

After a buffer solution is added to the donor stool in a suitablecontainer, the mixture should be blended to produce a slurry wherein thedonor stool is distributed substantially evenly within the buffersolution. The time spent blending may vary depending on the type ofblender used, but generally the slurry may be produced in approximatelyone (1) minute with a sterile electric blender.

In some embodiments, the resulting stool-diluent slurry may then undergoa form of filtration to separate the large fiber particles from thebacterial and other small molecule components of the stool material. Insome embodiments it may then be further concentrated into a microbialpellet. Filtration methods may include centrifugation, vacuumfiltration, sieve filtration using gravity, or other mechanisticfiltration methods.

In some embodiments, the filtration done by centrifugation may take atleast one centrifuge cycle and, in some cases, at least two (2)centrifuge cycles. The centrifuge may or may not be refrigerated,however, in preferred embodiments, the stool should generally be keptunder low temperature conditions. This may be accomplished by handlingthe stool on ice, storing product in a refrigerated space between steps,and in some embodiments, using a thermometer to ensure the stool is notexposed to temperatures above a certain threshold level.

A prepared fecal slurry undergoes at least two (2) centrifuge cycles, afirst cycle may be used to remove fibrous particulate from thestool-diluent slurry. Although any number of centrifuges may be suitablefor this step, an example of appropriate equipment is a Thermo-FisherSorvall ST 40 centrifuge suitable for use with 50 mL conical tubes. Ifusing such a centrifuge or similar, the slurry may be poured intoseveral 50 mL centrifuge conical tubes. In some embodiments, the slurrymay be centrifuged for approximately fifteen (15) minutes atapproximately 2000 RPM (827 G) or any other suitable RPM. The primarypurpose of the first centrifuge cycle is to separate a first fiberpellet from the supernatant, so the time and RPM may vary. The firstfiber pellet may be properly and sanitarily discarded, so that theremaining supernatant may be used in the second centrifuge cycle or forprocessing into the final product.

In one embodiment, after the first centrifuge cycle and subsequentdisposal of a particulate and fiber pellet, the remaining supernatantmay be poured into a second 50 mL conical vial, for example. The vialcan be centrifuged for a second cycle. The optional second cycle may runfor approximately twenty-five (25) minutes at 4,600 RPM (4613 G) or anyother suitable RPM. The second cycle is intended to compact theresulting microbial pellet. The RPM speed and time can vary. After thesecond cycle is complete, any remaining supernatant is removed from thevial. Unlike the first cycle, the microbial pellet resulting from thesecond cycle will be kept. The bacterial pellet can then be resuspended1:1 ratio with supernatant from the second centrifugation step. Thisliquid product can then alternately be encapsulated and frozen for oraldelivery or further lyophilized intro a stable powder form that isencapsulated for oral delivery or use in other embodiments (i.e.,topical).

Approximately for every about 1 to about 8 grams of fecal material (rawstool prior to the addition of a buffer or water), about 1 mL of liquidunlyophilized or about 100 mg of lyophilized powder product respectivelycan be obtained when using the methods described herein, for example, asset forth in Example 1.

Fecal Microbiota Compositions. Freezing may be accomplished in severalways, such as, but not limited to: flash freezing, in a regular freezer,or freeze-drying via sugar crystallization or lyophilization.Sterilization can occur before or after filtering of the fiber and largeparticulate. Sterilization can include microwave irradiation, ozone,pasteurization, autoclaving, HIUS, or sonication. Preferably,sterilization is performed by autoclaving.

When the filtered stool slurry or microbial pellet is freeze-driedbefore or after sterilization, the end-product is a sterilizedlyophilized or freeze-dried powder. This powder can then be used aloneor formulated into a topical application. A topical can include any oneor more of the following a screening agent, a vitamin, an essential oil,a plant protein, an anti-oxidizing agent, a preserving agent, afragrance, a ceramide, a moisturizing agent, a lubricating agent, apolysaccharide, and a filler.

It can also be formulated into a food or food product, nutraceutical,supplement, dietary supplement, or other form for oral ingestion. Theliquid or lyophilized FSP can be added to a beverage, including a sportsbeverage. It can be formulated for vaginal or rectal implantation or foradministration to a sinus passage via spray or gel.

The liquid or powder composition obtained by the described methods canbe emulsified with various emulsifying agents. For example, the liquidor powder can be emulsified with cetyl alcohol, stearyl alcohol,octacosanol, palmitic acid, stearic acid, and combinations thereof.

A liquid bacterial pellet suspension can be stabilized by lyophilizationor freeze-drying. Additional stabilizers can be added to thecomposition. A stabilizing filler may be added to the oral powder orencapsulated form, such as, but not limited to: collagen, glutamine,fiber (e.g., inulin, slippery elm, psyllium, chia, flax), orcarminatives (e.g., ginger, fennel, rose, licorice, peony). Astabilizing filler may also provide additional benefits to a recipientas a prebiotic, but additional benefits are not necessary as asterilized fecal composition may be the more important factor ineffective treatment of the patient.

A stabilized powder may then be deposited into at least one capsule ortablet layer, or otherwise encapsulated in any suitable enteric coating.For example, a sterilized fecal composition can be encapsulated in a No.1 gelatin capsule, followed by No. 0 and No. 00 capsules to create atriple encapsulated end product. However, not all capsules may be tripleencapsulated. Double or single encapsulation can also be used.Encapsulated formulations can be stored at approximately 20, 5, −20 to−80 degrees Celsius.

A single donor stool donation may be capable of creating numerous doses,which may then be used by a patient at the time of treatment.

Effective dose size may vary from as small as about 0.25-0.50 mL toabout 7.5-15 mL (and any 0.25 ml value in between the indicated range)of a sterilized unlyophilized frozen liquid fecal-derived composition.Effective dose sizes for lyophilized powder fecal-derived sterilecomposition may vary from as small as about 10 mg to about 5 g (and any1 mg value in between the indicated range).

3. Methods of Using Fecal Derived Sterilized Postbiotic Compositions

A subject's microbiota and the associated molecules of the microbiotaare foundational in interacting and signaling with the immune system,endocrine system, and nervous system. (Bordon 2019; Rooks 2016; Cani2016, Clarke 2014). The interaction of the microbiota and its associatedmolecules impacts the immune system of a subject and its performance. Asubject's microbiota can protect a subject from pathogens (Belkaid2017). When a subject's microbiota is out of balance, it is sometimesreferred to as dysbiosis of the microbiome (Petersen 2014, Weiss 2017).This dysbiosis or imbalance of a healthy microbiome ecosystem can allowopportunistic infections to thrive or contribute to inflammation andinappropriate immune response in a subject. These opportunisticinfections can additionally cause or exacerbate a disease or condition(Hand 2016).

Non-sterilized fecal microbiota transplant (FMT) therapy has provided aunique example of how microbial dysbiosis in the gastrointestinal (GI)tract can be improved in order to treat disease. In the past,non-sterilized FMT has been used to treat the opportunistic infectionClostridium difficile (CDI) associated with post antibiotic treatmentdysbiosis and recurrent CDI. Non-sterilized FMT has a significant amountof evidence demonstrating its efficacy, safety, and superiority of itsuse over other options for treating and ameliorating rCDI (Juul 2018,Wang 2016, Hvas 2018). Several randomized trials have shown thatnon-sterilized FMT is effective at resolving diarrheal symptoms in rCDI,while further preventing CDI recurrence with rates between 70-95% (Kelly2016, Cammarota 2015). Fecal matter differs between subjects.Additionally, there can be variation even between single donor samples,and the protocols for preparation and delivery of non-sterilized FMTalso can substantially differ. However, all of the non-sterilized FMTpreparation and delivery methods (oral encapsulated, enema, colonoscopy,or a nasojejunal/nasogastric tube) have demonstrated similar efficacywithin the general population (Kao 2017, Lee 2016, Ramal 2019, Zhang2019). Clinical data has shown that autologous, freeze-dried, and fecalfiltrate with phage only as preparations of non-sterilized FMT are alsoeffective in treating rCDI with similar efficacy to fresh and frozenpreparations (Kelly 2016, Staley 2017, Ott 2017).

The success of using non-sterilized FMT to treat rCDI, and furtherunderstanding of the microbiota's role in human physiology has led tonon-sterilized FMT being explored as a therapeutic option in many otherdisease states. Data from in vitro and in vivo research including over300 human clinical investigation new drug (IND) trials fornon-sterilized FMT has provided a large body of evidence that there isbroad therapeutic potential (Antushevich 2020). These studies haveprovided preliminary evidence of improved outcomes using non-sterilizedFMT in a variety of diseases ranging from obesity and metabolic diseaseto neuro-inflammation and neurodegeneration (Yu 2020, Wortelboer 2019).Some of the stronger evidence available has shown non-sterilized FMT canbe used with efficacy for IBD (Moayyedi 2015), Autism Spectrum Disorder(Kang 2017), hepatic encephalopathy (Bajaj 2019), and adjunctive supportfor immunotherapy in cancer (Chen 2019). However, the mechanismunderlying non-sterilized FMT's efficacy for any of these applicationsrequires further elucidation. The widely held assumption is that thereare important living bacterial species that impact health and increasethe possibility of engraftment post-transplant (Khoruts 2016, Wilson2019). The gastrointestinal tract of patients with many disease states,including rCDI, have been correlated with microbiome dysbiosis (Levy2017). The dysbiotic baseline of rCDI patients via bacterial sequencinghas been shown to improve after treatment with non-sterilized FMT (Kao2018, Smillie 2018). There have also been several studies that show ashift in the postbiotic molecules of the microbiota measured bymetabolomics in recipients of non-sterilized FMT correlating with thereturn to a healthy microbial profile (Nusbaum 2018, Martiniz-Gili2020).

A fecal-derived sterilized postbiotic composition as described hereincan be used to shift disease states from diseased to healthy whenadministered to a subject in a therapeutically effective dose. The shifttherefore is not linked to a bacterial species-specific mechanism thatoccurs through the transfer of a live or non-sterilized FMT.

As newly disclosed herein, a sterilized and filtered FMT comprisesvarious FMT metabolites. It has been theorized that FMT microbes canproduce 10²-10³ molecules that influence human physiology directly andindirectly. The FMT synthesized molecules are concentrated at theepithelial surfaces that directly interface with the microbiota,including the GI tract, skin, bladder, lungs, oral cavity, sinus cavity,and vagina.

The process provided here provides a sterile fecal-derived postbioticcomposition. Using this sterilized fecal-derived postbiotic composition,one can further extract or refine specific metabolites. A fecal-derivedsterilized postbiotic contains bacterial metabolites and other cellularcomponents. The sterilized fecal-derived postbiotic composition cannormalize an environment in a subject in need thereof by allowingexisting bacteria to colonize normally eventually supplanting adisease-associated dysbiotic colonization.

As demonstrated herein, the active components produced by the FMTsterilization process disclosed in successful applications of bacterialand fecal-derived products are not limited to live bacterial cells. Thefollowing summarizes metabolite effects in rCDI, beneficial effects ofheat-killed single strain probiotics upon oral administration, andeffects of differing administrative routes for other epithelial surfacessuch as the skin.

Two groups of bacterial derived metabolites that have been extensivelystudied and are suspected of playing a role in the mechanism of FMT'sability to treat rCDI are bile acids and short chain fatty acids(SCFAs). Primary bile acids are secreted by the liver and modified inthe gut by specific bacteria that have enzymes our human cells lack. Theelevation of primary bile acids in rCDI patients has been shown topromote the germination of the C. difficile spores (Sorg 2008). Incontrast, secondary bile acids that have been metabolized by specificbacteria are observed to increase after FMT treatment and correlate withsymptom resolution. Mechanistically secondary bile acids have been shownto inhibit the growth of the vegetative C. difficile bacteria(Thanissery 2017).

The SCFA valerate has also been shown to play a mechanistic role intreating rCDI by inhibiting the growth of the vegetative state of C.difficile in vitro and reducing C. difficile in mice when administeredto the mice orally (McDonald 2018). Both groups of molecules, SCFAs andsecondary bile acids, have been shown to be reduced in rCDI patientsrelative to healthy control subjects. Both SCFAs and secondary bileacids have been indicated to increase after non-sterilized FMT treatment(Allegretti 2016, Brown 2018, Weingarden 2014, Seekatz 2018). Specificbile acids and SCFA are also known to modulate the human immune systemby inducing T-regulatory cells (Smith 2013, Campbell 2020, Song 2020,Arpaia 2013).

There is a growing body of literature supporting the physiologicallyactive role of single-strain bacterially derived postbiotic molecules(Piqué 2019). A variety of mouse and human studies have explored theimpact of live versus heat-killed probiotics (Gao 2019, Wei 2019,Malagón-Rojas 2020). Pasteurized Akkermansia muciniphilia has been usedto investigate this concept. It was shown in a proof of conceptplacebo-controlled study to improve insulin sensitivity (p=0.002), whilereducing insulinemia in a population of 32 overweight/obese insulinresistant patients (Depommier 2019). Other species of heat-killedbacteria, lactic acid bacteria and Bifidobacterium have shown promisingimpacts on health through immune modulation and improving intestinalbarrier function (Chen 2013, Nakamura 2012, Thakur 2016, Sang 2015).Additionally, there have been several tissue culture studies exploringthe mechanisms that heat-killed bacteria can impact cancer cell growthin adenocarcinoma and colon cancer cell lines (Karimi 2019, Chung 2019).

The epithelial layer of the human body is covered in a microbialecosystem central to human health. Dysbiosis of the microbiota on any ofthe epithelial layer can impact the health of the subject epithelium.The compositions described herein of a fecal-derived sterile postbioticcan be used not only to treat the gastrointestinal tract but also treatinfections in the mouth, sinuses, and on the skin.

Sterilized FSP and lyophilized sterilized FSP can be used to formulatevarious products. Dosages of each respectively can be about 0.25 mL toabout 15 mL per day (and any 0.25 mL amount in between this range) andabout 10 mg to about 5 g per day of a lyophilized FSP (and any 5 mgamount in between this range) per subject.

The sterilized FSP and lyophilized sterilized FSP can be formulated asappropriate for administration orally, intranasally, vaginally,rectally, or on the skin. The sterilized FSP and lyophilized sterilizedFSP can be formulated into a capsule, soft gel, tablet, pill, gel,lotion, liquid or syrup, suppository, powder, mouthwash, and paste forpurposes of administration.

Methods of Use. A sterilized lyophilized or unlyophilized fecal-derivedpostbiotic composition can be used as an additive. Generally, anunlyophilized capsule is 0.5 mL of material and a lyophilized capsulecan contain about 50 mg to about 100 mg (and any 5 mg value in betweenthis range) In some embodiments about 12 mL to about 60 mL of liquidunlyophilized FSP can yield about 1 gram of lyophilized FSP powder.

A sterilized fecal composition can also be used as an adjunct with aprobiotic, synthetic bacterial community, or combined in a food ornutraceutical having a probiotic to augment probiotic function wheningested or otherwise administered to a subject. As low as 10 mg of ourlyophilized FSP per 1 gram or mL of probiotic mixture can be provided toa subject as an adjunct; however, larger amounts can be administered.

A sterilized fecal composition disclosed herein can also be used to beadministered with, after or before the administration of a livebiotherapeutic product (LBP) to augment its growth upon administrationto a subject. For example, the compositions described herein can beadministered with for example, Seres Therapeutics SERES-109, FinchTherapeutics CP101, Rebiotix RBX7455, Vendanta VE303, and similar LBPs.

A sterilized fecal-derived postbiotic composition can be used to treat acondition or disorder that is being treated with a non-sterilized FMT.The conditions and diseases contemplated for treatment include but arenot limited to the following Clostridium difficile infections, recurrentC. difficile infections, a C. difficile related condition, a mooddisorder, fatigue, an autism spectrum disorder (ASD), inflammatory bowelsyndrome constipation (IBS-C), inflammatory bowel syndrome diarrhea(IBS-D), inflammatory bowel disease (IBD), a subject who has receivedimmunotherapy to treat a cancer, a metabolic syndrome, aneurodegenerative disease, and dysbiosis. A metabolic syndromecontemplated for treatment includes a cluster of biochemical andphysiological abnormalities associated with the development ofcardiovascular disease, stroke, and type 2 diabetes (Grundy 2004).Components of metabolic syndrome related to cardiovascular disease andconsidered for treatment or amelioration with the described compositionsinclude abdominal obesity, atherogenic dyslipidemia, raised bloodpressure, insulin resistance with or without glucose intolerance,proinflammatory state, and prothrombotic state.

Stool Processing for Metabolite Extraction. A sterilized fecalcomposition contains metabolites. Metabolites are preserved throughoutthe process from raw stool to a sterilized product. For example,retention of short chain fatty acids (SCFAs) after lyophilization wasshown to be pH-dependent. Increasing the pH of an FSP product prior tolyophilization resulted in an increased retention of SCFA. The fact theSCFAs were retained in any concentration during the lyophilizationprocedure was unexpected due to conventional understanding anddefinition of SCFA being volatile compounds (Rios-Covian 2016). Thus, apreferred method of preparing a sterilized and lyophilized fecalcomposition comprises increasing the pH of the composition to a pH ofabout 6.0 to about 9.0, or about 7.2 to about 7.8 (and any 0.1 value inbetween these ranges) prior to lyophilizing the composition. It was alsounexpected that metabolites were retained across metabolite classesafter both sterilization by autoclaving and lyophilization.

Short chain fatty acids (SCFA) are a well-studied class of bacterialderived secondary metabolites in postbiotics. SCFAs are produced as anend-product of anaerobic fermentation of complex carbohydrates by themicrobiota in the colon (Wong 2006). Acetate and butyrate are essentialfor healthy human physiology because they are a primary energy sourcefor colonocytes. Propionate, the other most abundant SCFA, is utilizedin the liver as a substrate for glucose production (gluconeogenesis)(den Besten 2013). The SCFA acetate, butyrate and propionate ratio canbe generally represented in a ratio of 60:20:20 (Bergman 1990, Cummings1987). The ratio is further described infra in Example 2. SCFA have alsobeen shown to interact with a wide array of human cells and impact theimmune system and metabolism throughout the body via G protein-coupledreceptors (GPCRs). In addition, butyrate has been shown to act as a HDACinhibitor which impacts the epigenetics of human cells independent ofGPCRs (Chang 2014). These important postbiotics are not only conservedfrom the stool product throughout our process, but their concentrationsare slightly increased after the sterilization process step ofautoclaving; the relative ratios are conserved as discussed in Example2. The method of producing the sterilized fecal-derived postbiotic canbe further enhanced by increasing the pH prior to lyophilization, asdemonstrated in the examples. Valerate is an important SCFA and ismaintained at an estimated concentration of 200-800 μg/g in thesterilized lyophilized FSP product compared to concentrations of 18-45μg/g valerate obtained using a traditional FMT procedure.

Another class of metabolites present in postbiotics are the bile acids.There is evidence that several secondary bile acids including the onesidentified using metabolomics analysis act as immunomodulators that caninduce Tregs instead of Th17 (Campbell 2020, Song 2020, Hang 2019). Thesecondary bile acid, ursodeoxycholic acid, has long been used in thetreatment of liver disease. Several synthetic bile acids are pipelineblockbuster drugs for NASH and NAFLD (Hofman 1989, Neuschwander-Tetri2015). Taurodeoxycholic acid (TDCA) is another secondary bile acid thathas been shown to impact immune function. In a mouse model TDCA was usedintravenously to treat septicemia by increasing granulocyticmyeloid-derived suppressor cells (Chang 2018). The sterilization andlyophilization method disclosed herein maintained the composition ofsecondary bile acids and revealed a significantly increasedconcentration of taurodeoxycholate specifically. See FIGS. 4 and 5 .

Another metabolite present in the sterilized FSP products are tryptophanmetabolites. Tryptophan metabolites have been studied for their role inneuroimmunomodulation and inflammatory signaling (Ye 2020, Bansal 2010),Indole derivatives have been shown to bind the aryl hydrocarbon receptorwhich has a broad array of functions including impacting immuneactivation (Rothhammer 2019). Indole-3-propionic acid contributes toremission through an IL-10 mediated immune pathway in human and mousemodels of colitis (Ale 2018). This class of molecules is conserved inthe sterilization method described herein.

Ceramides and sphingosines have been well established to play a role inskin moisture retention (Geilen 1997). Newer evidence suggest they playa role in immune signaling and cell turnover in keratinocytes (Uchida2014, Jeong 2015). The methods of producing the described sterilized FSPproduct contains a variety of ceramides and sphingosines within thefatty acid profile of our FSP product as reflected in the examples.

EXAMPLES

The following sections describe selected aspects of exemplary systemsand methods for performing FSP treatment. The examples in these sectionsare intended for illustration and should not be interpreted as limitingthe entire scope of the present disclosure. Each section may include oneor more distinct inventions, functions, and/or steps.

Example 1

A method of preparing a sterilized fecal derived postbiotic is obtainedas follows. Screened healthy donors are identified and enrolled. Donorscollect stool and freeze at −20° C. immediately after bowel movement.Stools are then transported to the lab for processing. The stool isprocessed as follows. The stool donor identification is recorded withthe total stool volume, the Bristol stool scale for donation, the timeof stool donation, and the date of the stool donation. The frozen stoolis removed from the freezer. The stool is allowed to thaw for up to 24hours in the refrigerator. The stool is measured out into 50 to 150 gportions of stool and placed into an autoclaved blending jar. The amountof stool in grams is weighed. Distilled water is then added to the stoolat a ratio of 5:1 based on the stool mass to blending jar (e.g., 500 mLof distilled water is added to 100 g of stool.) The amount of distilledwater is recorded.

The stool and distilled water are blended for 60 seconds. The fecalcontaining slurry is then poured into single use 50 mL centrifugeconical tubes, with each conical containing 50 mL of the slurry.

Conical tubes are centrifuged for a first run for 15 to 20 minutes at2000 RPM (872G) in a Thermo-Fisher Sorvall ST 40 centrifuge withswinging bucket rotor TX-750 using 50 mL conical inserts.

The conical tubes are removed after centrifugation and the supernatantpoured into new sterile 50 mL conical tubes. Volume is re-measured foreach of the tubes. The pellet in the initial tubes containing the largeparticulate and fiber is discarded. The conical tubes containing thecentrifuged supernatant is centrifuged a second time for 25 minutes at4600 RPM (4613G). The conical tubes are then removed from the centrifugeand the supernatant is poured into a sterile glass container for uselater. The pellet remaining in the tubes from the second centrifugationis resuspended using a vortex and transfer pipette method. Theresuspended pellets is homogenize. Total volume of the microbial pelletis measured. The pellet is then resuspended with a one to one ratio withthe previously saved supernatant from the second centrifugation.

The pellet supernatant mixture is poured into a glass jar with aself-sealing lid, labeled with autoclave tape containing donor ID andsample collection date, and then autoclaved. The samples are autoclavedfor 30 minutes at 121° C. Samples are then removed from the autoclave.Sterility is verified when the autoclave tape shows completion. Thisverifies time, pressure, and temperature to assure sterility. The jarsare placed in a refrigerator to cool. This liquid represents thefecal-derived sterile product referenced in further examples as liquidFSP.

The liquid FSP product can additionally be further stabilized. Theproduct is removed from the refrigerator after cooling and the pHmeasured. The pH is readjusted to 7.2 or higher (no higher than 9.0)using NaOH. The FSP liquid is poured into autoclaved lyophilizingglassware. Total volume by weight is measured. The benefit of increasingthe pH prior to lyophilization is illustrated in the data of FIG. 3 .

The lyophilizing glassware containing sterilized FSP product is thenflash frozen in a 90% isopropyl alcohol dry ice bath. The jars are thenlyophilized for 4 to 14 hours at <0.3 mBarr. Once dry, the jars areremoved from the lyophilizer or and the dry weight of the powder ismeasured. The lyophilized powder is then placed in an autoclaved glassjar for storage. These jars are stored at room temperature or −20° C.(the freezer is lower humidity) in the dark. Alternatively, the powdercan be placed in dark glass jars for storage.

Example 2

The method of obtaining the fecal-derived sterilized postbiotic liquid(FSP) and lyophilized powder (FSP-L) is as described in Example 1. Theliquid FSP and FSP-L were analyzed for quantitative and relativeconcentrations of metabolites. Multiple donors and stools from differenttime points were analyzed to elucidate metabolomic profile differences.In addition, FMT samples, as controls, were collected immediately priorto the autoclaving as described in Example 1 for each sample analysis(referred in all figures as “FMT”). Metabolomic analysis was performedMetabolon, Inc., Morrisville, N.C., USA, by using a global untargetedUPLC-MS/MS platform and a specific LC-MS/MS-based targeted assayspecifically to quantify short chain fatty acids (SCFA). Analysis ofseveral groupings of samples from the raw data from Metabolon wasperformed using MetaboAnalyst software for normalization and statisticalanalysis.

FIG. 1 shows the concentration of various short chain fatty acids (SCFA)metabolites found in fresh processed versus frozen processed accordingto Example 1 at the three time points; FMT, FSP and FSP-L from a singledonor stool. The freezing of the stool prior to processing did reducethe total SCFA concentrations a little. But, it was unexpected thatthere was still a retention of the SCFA with the frozen process withmaintained relative ratios, validating that this process would be afeasible alternative to the conventional model of processing fresh stoolfor FMT. FIG. 2 compares the concentration of SCFAs present in rawstool, unsterilized FMT, sterilized fecal derived probiotic that is notlyophilized (FSP), and lyophilized FSP (FSP-L). The unsterilized FMT isobtained immediately prior to the autoclave step in Example 1. The FSPand lyophilized FSP (FSP-L) were obtained by processing the stool asoutlined in Example 1. All four samples were from the same donor'sstool. Unexpectedly, processing the stool according to Example 1resulted in an increase of SCFA concentration after the autoclavingsterilization step and much less loss of SCFAs from the lyophilizationstep than expected as well. It was assumed that all SCFAs wouldvolatilize during the lyophilization process. The unexpected increase ofthe SCFAs after autoclaving may be due to the heating or releasing ofSCFA from inside cells during the lysis process. The retention of theSCFA during lyophilization may be due to the basic pH of the samples asoutlined in FIG. 3

The ratio of short chain fatty acids (SCFAs) remains unchanged atapproximately 60:20:20 for acetic acid, propionic acid and butyric acidrespectively for unsterilized FMT samples, the unlyophilized FSP sample,and the lyophilized FSP sample (FSP-L). This is significant because ofprior publications that discuss the physiologic relevance of the SCFAratios discussed above. It is also unexpected because we anticipatedsignificant loss of SCFA and not uniform maintenance of the ratiosthroughout the processing.

Sample Acetic Acid Propionic Acid Butyric Acid ID Donor Type mM mM mM A1 FMT 10.2 3.1 3.6 B 1 FSP 13.3 3.8 4.0 D 2 FMT 9.6 2.7 4.0 E 2 FSP 9.73.0 4.5 G 2 FSP-L 5.7 1.7 2.8 I 3 FMT 5.6 2.7 1.5 K 3 FSP 7.2 3.2 1.9 M3 FSP-L 4.8 2.2 1.2

Additionally, we observed an increase in the relative concentration ofthe secondary bile acid taurodeoxycholic acid (TDCA) as a result of theprocess to produce FSP from FMT as depicted in FIG. 5 . The observedincrease in the relative concentration of TDCA was unexpected.

Presence and relative quantity of lipid components were assessed;however, absolute concentration was not determined. Lipid componentspresent in all FMT, FSP, and lyophilized FSP (FSP-L) samples that wereanalyzed included: long chain monounsaturated fatty acid, hydroxyl fattyacid, dicarboxylate, acyl glycine, mono unsaturated acyl carnitine, acylcholine, dihydroxy fatty acid, phosphatidylcholine (PC),lysophospholipid, diacylglycerol, ceramides, lactosylceramides (LCER),long chain saturated fatty acids, long-chain polyunsaturated fatty acids(n3 and n6), branched fatty acids, polyunsaturated acyl carnitine, longchain saturated acyl carnitine, monohydroxy fatty acid, endocannabinoid,phosphatidylethanolamine (PE), lysoplasmalogen, monoacylglycerol,galactosyl glycerolipids, dihydroceramides, hexosylceramides (HCER), anddihydrosphingomyelins. These components were identified via theMetabolon global metabolomics LC/MS/MS and Polar LC platform.

In another assay, the metabolites were compared and statisticallyanalyzed between FMT and sterilized fecal-derived postbiotic (FSP)steps. This analysis was done for stools processed from the same donorand different donors as obtained in Example 1 with a p<0.1. It wasunexpected that of the data analyzed, there were no metabolites with ap<0.05 between FMT and FSP steps from different donors. That is why FIG.6 is presenting metabolites with a p<0.1 as a comparator of the FMT toFSP from a single donor and different donors. The assay was performed byMetabolon using global metabolomics mass spectrometry analysis and thenthe data was statistically analyzed using MetaboAnalyst software asdiscussed in Example 1. This comparative analysis identified onlythirty-two (32) metabolites from 853 named metabolites that change witha p<0.1 regardless of whether the comparison of FMT to FSP was doneusing three stools from a single donor or three unique donor stools;these metabolites are sensitive to thermal change or thermaldegradation. The 32 metabolites that change are: N-acetylmethioninesulfoxide; glutamine; S-carboxyethylcysteine; 1-linoleoyl-GPE (18:2);3-hydroxylaurate; 3,4-dihydroxybenzoate; cyclo(gly-pro);guanosine-2′,3′-cyclic monophosphate; oxindolylalanine;3-hydroxymyristate; cryptochlorogenic acid; formiminoglutamate;3-hydroxytridecanoate; 1-linoleoyl-GPG (18:2); uridine-2′,3′-cyclicmonophosphate; cytidine 2′,3′-cyclic monophosphate; cytidine;cyclo(pro-tyr); 4-vinylcatechol sulfate; sucrose; nicotinamide;nicotinamide riboside; 2,4-dihydroxybutyrate; propionylglycine (C3);cytidine 2′ or 3′-monophosphate (2′ or 3′-CMP); cyclo(his-pro); maltol;N-acetylmuramate; 3-ketosphinganine; 1-palmitoylglycerol (16:0);3-amino-2-piperidone; and glutamate gamma-methyl ester. See FIG. 6 Thisvenn diagram outlines that although some metabolites are impacted by theheating process of autoclaving. Most are not and the overall metabolomiccomposition is retained.

Example 3

According to one example, a 34-year-old female presenting with fatigue,constipation, anxiety, and depression was treated with capsulescontaining fecal-derived sterilized postbiotic product of one 0.5 mLfrozen liquid FSP capsule daily for 30 days. The patient reportedsignificant improvement in constipation and fatigue after the first 30days. The patient self-reported that her energy increased from a 4 to a7/10 and that her constipation went from multiple days of no stool, topassing stool 6-7 days each week. The patient continued taking 0.5 mLfrozen liquid FSP capsules having sterilized fecal composition for anadditional 5 months. The patient's energy ratings scale increased to8.5/10 after the second month. In addition to physical complaints by thepatient, mental health was assessed using validated scales including thePHQ-9 and the GAD-7. The patient's initial score was 13 when measured ather initial intake in February indicating “moderate depression.” Sevenmonths later, after continuous dosing, the patient's follow-up PHQ-9score improved to one. The patient's baseline GAD-7, which is a metricfor anxiety, was a 3 prior to treatment with the sterilized fecalpostbiotic composition; upon follow-up, her GAD-7 dropped to 0.

Example 4

In another example, a 70-year-old male having rCDI was treated. Thepatient presented after a second recurrence of a C. difficile infection,while undergoing treatment with a Vancomycin taper, which improved hisdiarrhea symptoms but did not completely resolve the condition. Thepatient was treated with a fecal derived sterilized postbiotic asobtained by the method of Example 1. The subject was treated withcapsules containing the fecal-derived sterilized postbiotic that weredosed as 10 capsules for 3 days, and 5 capsules for 6 additional days inconjunction with continued Vancomycin taper. Each capsule containedapproximately 0.5 mL frozen liquid of the composition.

The patient reported complete resolution of diarrheal symptoms andsignificant improvement in fatigue after starting the FSP. Theseimprovements were not present during his first week of Vancomycintherapy or previous treatments.

Example 5

A 17-year-old male presented with a history of PANDAS, Autism SpectrumDisorder and Anxiety/OCD. The patient's primary symptoms at initialvisit were OCD, brain fog, and anxiety. He was treated with one 0.5 mLfrozen liquid FSP capsule daily for 6 months, of the capsules describedas obtained in the example above. The patient reported significantimprovement in brain fog and obsessive-compulsive disorder (OCD)symptoms within the first week of treatment at an initial check in atone month. He continued to have improvement and each month requestedadditional capsules. At a follow up visit at 6 months, the patient notedthat he continued to have improvement in symptoms of brain fog and OCD.He also reported at six months that he had no acute episodes ofinflammation that in his previous medical history resulted in treatmentwith intravenous immunoglobulin (IVIG).

Example 6

A 69-year-old male with a history of rCDI was previously treated withfecal transplant capsules. The patient returned a year later with theclinical symptom picture of rCDI without confirmatory labs. The patientwas treated with the fecal-derived sterilized postbiotic compositionobtained as described in Example 1. The capsules were dosed at 10-0.5 mLfrozen capsules daily for 3 days, followed by one capsule daily for 18days. He was feeling well until he experienced an adverse reaction to anover-the-counter (OTC) non-steroidal anti-inflammatory drug (NSAID)consisting of diarrhea. This provoked the need for a one time, 5capsules loading dose, followed by 1 capsule daily for 7 days. Thepatient reported complete resolution of diarrheal symptoms andsignificant improvement in fatigue at the one-month follow-up. Diarrhealsymptoms remained resolved at a 4-month follow-up and there was no CDIrecurrence.

The different embodiments of compositions and methods described hereinrelate to a fecal-derived sterilized postbiotic composition thatprovides several advantages over previous systems and methods. Thecompositions provide enhanced safety and efficacy over unsterilized FMTtherapies. The compositions minimize the risk of transmitting infectionfrom donor stool to a recipient of an FSP capsule or batch of capsules.Furthermore, the illustrative embodiments may provide a way to mitigatethe health risks associated with using live bacteria, withoutsacrificing effectiveness of the treatment. Specifically, systems andmethods disclosed herein enable the safe delivery of fecal-derivedsterilized postbiotic composition and metabolites contained therein toany epithelium of a subject in need.

Thus, the illustrative embodiments described herein are particularlyuseful for patients who desire the potential benefits of FMT therapies,while minimizing detrimental side effects associated with conventionalmethods. However, not all embodiments described herein provide the sameadvantages or the same degree of advantage.

Embodiments

Embodiments of the compositions, methods of making the compositions andmethods of using the composition include for example the following:

-   -   [1] A method of making a sterilized fecal derived postbiotic        composition comprising:        -   (a) blending a stool sample from a subject with a buffer;        -   (b) removing particulate and fibrous matter from the blended            stool sample to obtain a liquid; and        -   (c) sterilizing the liquid to produce the sterilized fecal            derived postbiotic composition.    -   [2] The method of claim [1] further comprising a step of        stabilizing the sterilized fecal derived postbiotic composition        by lyophilization.    -   [3] The method of claim [2], wherein the stool sample is frozen        when collected.    -   [4] The method of claim [2], wherein the pH of the sterilized        fecal derived postbiotic composition is adjusted to about 6.0 to        about 9.0 prior to lyophilization.    -   [5] The method of any of claims [1 to 4], wherein the stool        sample is derived from a healthy screened donor.    -   [6] The method of any of claims [1 or 5], wherein the        sterilizing step is by autoclaving.    -   [7] The method of claim [1], wherein the step of sterilizing is        performed prior to step (a) or step (b).    -   [8] The method of claim [6], wherein the autoclaving is        performed at about 121 to 134° C. for about 30 minutes or at        about 135 to 140° C. for about 20 minutes.    -   [9] The method of claim [1], wherein the step of blending is        accomplished by the use of a comminution device selected from        the group consisting of: a crushing device, a grinding device,        and a homogenization device.    -   [10] The method of claim [1], wherein the buffer is a phosphate        buffered saline solution, maldextrin-trehalose solution, or        water.    -   [11] The method of claim [1], wherein the step of removing        particulate and fibrous matter is accomplished by a method        selected from the group consisting of: centrifugation,        filtration, membrane filter press drying, or a combination        thereof.    -   [12] The method of claim [1 or 11], wherein the removing step is        a multi-step process.    -   [13] A sterilized fecal-derived postbiotic composition        comprising one or more of short chain fatty acids, bile acids,        amino acid derivatives, ceramides, lipopolysaccharides, capsular        polysaccharides, and sphingosines.    -   [14] The sterilized fecal derived postbiotic composition of        claim 13, wherein the short chain fatty acid comprises formic        acid or one of its salts and/or one of its esters, acetic acid        or one of its salts and/or one of its esters, propionic acid or        one of its salts and/or one of its esters, butyric acid or one        of its salts and/or one of its esters, isobutyric acid or one of        its salts and/or one of its esters, valeric acid or one of its        salts and/or one of its esters, or isovaleric acid or one of its        salts and/or one of its esters.    -   [15] The composition of any of claims [13 to 14], wherein the        composition comprises a ratio by weight of acetic acid:propionic        acid:butyric acid respectively of between 2:2:1 and 3:1:1.    -   [16] The composition of claim [13], wherein the composition        comprises a ratio by weight of acetic acid:propionic        acid:butyric acid respectively of 60±5:20±5:20±5.12.    -   [17] The composition of any of claims [13 to 16] further        comprising a co-emulsifying agent, wherein the co-emulsifying        agent is selected from the group consisting of: cetyl alcohol,        stearyl alcohol, octacosanol, palmitic acid, stearic acid, and        combinations thereof.    -   [18] The composition of any of claims [13 to 17], further        comprising at least one component selected from the group        consisting of: a screening agent, a vitamin, an essential oil, a        plant protein, an anti-oxidizing agent, a preserving agent, a        fragrance, a ceramide, a moisturizing agent, a lubricating        agent, a polysaccharide, and a filler.    -   [19] The composition of any of claims [13 and 18], wherein the        composition is in the form of a solid, an aerosol, a pill, a        capsule, a tablet, a paste, a powder, a gel, a lotion, a liquid,        or a body wash.    -   [20] The composition of any of claims [13 to 19], wherein the        composition is admixed with a probiotic, a live biotherapeutic,        a synthetic microbial community, or a non-sterilized fecal        composition.    -   [21] A method of treating or ameliorating a condition in a        subject comprising:        -   administering to the subject (i) a composition of any of            claims 13 to 20, or (ii) a sterilized fecal derived            postbiotic composition comprising one or more of short chain            fatty acids, bile acids, amino acid derivatives, ceramides,            and sphingosines in a therapeutically effective amount,        -   wherein the condition is selected from the group consisting            of a Clostridium difficile infection, C. difficile related            condition, a mood disorder, fatigue, autism spectrum            disorder (ASD), inflammatory bowel syndrome constipation            (IBS-C), inflammatory bowel syndrome diarrhea (IBS-D),            inflammatory bowel disease (IBD), a subject who has received            immunotherapy to treat a cancer, a metabolic syndrome, and            dysbiosis.    -   [22] The method of claim [21], wherein the mood disorder is        anxiety, depression or obsessive-compulsive disorder.    -   [23] The method of any of claims [21 to 22], wherein the        sterilized fecal derived postbiotic composition is administered        to the subject with a non-sterilized fecal composition, live        biotherapeutic, and/or a probiotic.    -   [24] The method of any of claims [21 to 23], wherein the        sterilized fecal derived postbiotic composition is administered        orally, topically, intranasally, rectally, vaginally or        intravenously.    -   [25] An additive for a culture media for culturing probiotics or        live biotherapeutics comprising the composition of any of claims        [13 to 20].    -   [26] A dietary supplement, food or beverage comprising the        composition of any of claims [13 to 20].

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1. A method of making a sterilized fecal derived postbiotic compositioncomprising: (a) blending a stool sample from a subject with a buffer;(b) removing particulate and fibrous matter from the blended stoolsample to obtain a liquid; and (c) sterilizing the liquid to produce thesterilized fecal derived postbiotic composition.
 2. The method of claim1 further comprising a step of stabilizing the sterilized fecal derivedpostbiotic composition by lyophilization.
 3. The method of claim 2,wherein the stool sample is frozen when collected.
 4. The method ofclaim 2, wherein the pH of the sterilized fecal derived postbioticcomposition is adjusted to about 6.0 to about 9.0 prior tolyophilization.
 5. The method of any of claims 1 to 4, wherein the stoolsample is derived from a healthy screened donor.
 6. The method of any ofclaim 1 or 5, wherein the sterilizing step is by autoclaving.
 7. Themethod of claim 1, wherein the step of sterilizing is performed prior tostep (a) or step (b).
 8. The method of claim 6, wherein the autoclavingis performed at about 121 to 134° C. for about 30 minutes or at about135 to 140° C. for about 20 minutes.
 9. The method of claim 1, whereinthe step of blending is accomplished by the use of a comminution deviceselected from the group consisting of: a crushing device, a grindingdevice, and a homogenization device.
 10. The method of claim 1, whereinthe buffer is a phosphate buffered saline solution, maldextrin-trehalosesolution, or water.
 11. The method of claim 1, wherein the step ofremoving particulate and fibrous matter is accomplished by a methodselected from the group consisting of: centrifugation, filtration,membrane filter press drying, or a combination thereof.
 12. The methodof claim 1 or 11, wherein the removing step is a multi-step process. 13.A sterilized fecal-derived postbiotic composition comprising one or moreof short chain fatty acids, bile acids, amino acid derivatives,ceramides, lipopolysaccharides, capsular polysaccharides, andsphingosines.
 14. The sterilized fecal derived postbiotic composition ofclaim 13, wherein the short chain fatty acid comprises formic acid orone of its salts and/or one of its esters, acetic acid or one of itssalts and/or one of its esters, propionic acid or one of its saltsand/or one of its esters, butyric acid or one of its salts and/or one ofits esters, isobutyric acid or one of its salts and/or one of itsesters, valeric acid or one of its salts and/or one of its esters, orisovaleric acid or one of its salts and/or one of its esters.
 15. Thecomposition of any of claims 13 to 14, wherein the composition comprisesa ratio by weight of acetic acid:propionic acid:butyric acidrespectively of between 2:2:1 and 3:1:1.
 16. The composition of claim13, wherein the composition comprises a ratio by weight of aceticacid:propionic acid:butyric acid respectively of 60±5:20±5:20±5.12. 17.The composition of any of claims 13 to 16 further comprising aco-emulsifying agent, wherein the co-emulsifying agent is selected fromthe group consisting of: cetyl alcohol, stearyl alcohol, octacosanol,palmitic acid, stearic acid, and combinations thereof.
 18. Thecomposition of any of claims 13 to 17, further comprising at least onecomponent selected from the group consisting of: a screening agent, avitamin, an essential oil, a plant protein, an anti-oxidizing agent, apreserving agent, a fragrance, a ceramide, a moisturizing agent, alubricating agent, a polysaccharide, and a filler.
 19. The compositionof any of claims 13 and 18, wherein the composition is in the form of asolid, an aerosol, a pill, a capsule, a tablet, a paste, a powder, agel, a lotion, a liquid, or a body wash.
 20. The composition of any ofclaims 13 to 19, wherein the composition is admixed with a probiotic, alive biotherapeutic, a synthetic microbial community, or anon-sterilized fecal composition.
 21. A method of treating orameliorating a condition in a subject comprising: administering to thesubject (i) a composition of any of claims 13 to 20, or (ii) asterilized fecal derived postbiotic composition comprising one or moreof short chain fatty acids, bile acids, amino acid derivatives,ceramides, and sphingosines in a therapeutically effective amount,wherein the condition is selected from the group consisting of aClostridium difficile infection, C. difficile related condition, a mooddisorder, fatigue, autism spectrum disorder (ASD), inflammatory bowelsyndrome constipation (IBS-C), inflammatory bowel syndrome diarrhea(IBS-D), inflammatory bowel disease (IBD), a subject who has receivedimmunotherapy to treat a cancer, a metabolic syndrome, and dysbiosis.22. The method of claim 21, wherein the mood disorder is anxiety,depression or obsessive-compulsive disorder.
 23. The method of any ofclaims 21 to 22, wherein the sterilized fecal derived postbioticcomposition is administered to the subject with a non-sterilized fecalcomposition, live biotherapeutic, and/or a probiotic.
 24. The method ofany of claims 21 to 23, wherein the sterilized fecal derived postbioticcomposition is administered orally, topically, intranasally, rectally,vaginally or intravenously.
 25. An additive for a culture media forculturing probiotics or live biotherapeutics comprising the compositionof any of claims 13 to
 20. 26. A dietary supplement, food or beveragecomprising the composition of any of claims 13 to 20.